DE10257423A1 - Microscope used in molecular biology comprises a focussing arrangement producing an extended planar object illumination region, a detection device, and a movement arrangement - Google Patents

Abstract

Microscope (100) with at least one illumination beam path (2) and at least one detection beam path (5) comprises a focussing arrangement (3) for each illumination beam path for producing an extended planar object illumination region in the direction of and perpendicular to an illumination axis of the illumination beam path. A detection device (10) of the detection beam path is arranged approximately orthogonally to the planar object illumination region. A movement arrangement is provided for producing relative movement between the planar object illumination region and an object (4) to be investigated. Independent claims are also included for alternative microscopes.

Description

The present invention relates to a microscope according to the preamble of claim 1.

In contrast to working on individuals Cells are light microscopic examinations on embryos and other biological samples with special problems absorption and loss of resolution afflicted. For example, you can biological issues related to gene expression patterns in developing organisms currently difficult with light microscopic Imaging procedures are answered because these are often too slow, too low resolution or are technically complex or from the free working distance or from the Sample holder from an observation of millimeter-sized objects do not allow. An acceptable solution must be to handle large samples and a fast, high resolution Allow recording of the data and technically as possible be easy to implement.

From the scientific literature is a microscope for Oceanographic research is known for this is that there is an illuminating light plane in a sample chamber with a laser generated and perpendicular to this plane with a camera in the Illuminated light plane detected fluorescence signals detected [E. Fox et al., Opt. Express 10, 145 (2002)]. This microscope is similar to that Ultramicroscope by H. Siedentopf and R. Zsigmondy [Ann. Phys. 10 (4) 1 (1903)] and is used for the detection of individual free-floating particles such as bacteria used. It is not for that suitable, millimeter-sized, For example, to take samples of developmental biology, as a cell as Sample holder. Likewise, it is not for three-dimensional recordings suitable as there is no possibility features, to move the sample relative to the illuminating light plane.

From the DE 19720513 A1 or the US 5,903,781 as well as from the scientific literature [D. Huber et al., J. Microsc. 202, 208 (2001)], an instrument for three-dimensional macrography is known in which an arrangement for generating light planes is used for the photographic detection of objects. An object is moved through an illumination plane and the reflected and scattered light is detected with a camera. This device is used to make three-dimensional reconstructions of centimeter-sized objects. However, it is not suitable for the use of fluorescence signals or for the high-resolution reproduction of the objects. A slit figure aperture is used in connection with a mirror arrangement for the generation of the light planes. The sample cannot be rotated by using a sample table that only moves linearly, so that the sample cannot be observed from several sides.

Furthermore, from the technical-scientific Literature constructions for known optical tomography. Optical projection tomography is used, for example, in gene expression analysis [J. Sharpe et al., Science 296, 541 (2002)]. It refers to a system in which projections of biological samples are recorded with the sample around an axis perpendicular to the detection direction is rotated. Since the sample is not perpendicular to the detection axis the microscope has an illuminating light plane selectively illuminated in contrast to the microscope according to the invention a very big one Depth of field, through which a great Part of the sample recorded becomes. Therefore, the microscope does not offer the possibility of moving the sample along the detection axis to take a three-dimensional image. A three-dimensional Image of the sample with spatial resolution is therefore only possible through the reconstruction based on the projections.

From the DE 43 26 473 C2 a confocal theta microscope is known which is characterized in that it uses a first objective for point illumination and a second objective for imaging the object light onto a point detector, the detection direction being approximately perpendicular to the direction of illumination. As a result, the confocal overlapping area of the illumination volume with the detection volume is particularly small and the microscope achieves an almost isotropic resolution, the magnitude of which corresponds to the lateral resolution of a confocal microscope.

However, this theta microscope is arranged confocally, which places high demands on the relative adjustment of the lighting and detection focal points. Besides, is it despite a big one Working distance not easily able to take pictures of large objects close. This is because the object is in the theta microscope does not have enough freedom of movement when scanning objects and that it can be scanned in three directions because of point detection must what a recording takes a long time. The illuminating light becomes one Illumination point focused.

The basis of the present invention The task is to propose a microscope for high-resolution three-dimensional Observation of millimeters biological objects is suitable, with a quick absorption of the Data possible and the structure is technically possible is easy to implement.

This object is achieved by the microscope specified in claim 1. The sample is illuminated by a thin strip of light and the observation is perpendicular to this object lighting area, which has an area-like extension. The thickness of the illuminating light strip thus largely determines the depth of field of the system. For the image acquisition, the object is moved by the spatially fixed light strip, and fluorescent or / and scattered light are recorded in any position of the raster movement with a flat detector. Since the object can be rotated in the preferred embodiment, it is possible to take such three-dimensional images from several sides and to combine them into a single three-dimensional image, the resolution of which is only determined by the lateral resolution of the individual images. The high resolution of this image is the result of focused lighting, vertical detection, the movement of the object and the combination of the individual images through image processing.

The microscope according to the invention has a Illumination light path and a detection light path in the object lighting area are preferably orthogonal to each other, which means the detection direction is perpendicular to the illumination light level. However, the Sufficient advantages of the invention still achieved when the angle between the direction of illumination and the direction of detection or between the illuminating light plane and the detection direction in not too big Dimensions of deviates from a right angle.

It is advantageously used as a light source a laser is used that selectively excites fluorescence emission enabled in the sample. For focusing the illuminating light into a thin strip a cylindrical lens is preferably used, but it can also another focusing element (e.g. a holographic element or a conical lens (Axicon) or a phase plate or others Elements for generating a Bessel beam) are used.

The detected light is preferred Fluorescent light. Possible is also the detection of stray light. The detection light is preferably based on a telecentric system consisting of two lenses imaged the detector. However, other optical ones are also suitable Assemblies.

The detection is preferably carried out with a flat Detector that detects the entire field, for example a CCD camera. By using such a detector, a quick image acquisition is possible possible, and the movement of the sample for a three-dimensional image is in one direction (namely along the Detection axis) limited. The resolution of the system is determined by the lateral resolution of the detection optics.

Because the area of the detectors currently available generally not sufficient for a complete, high-resolution recording of several millimeters in size To ensure objects exists in one embodiment of the microscope according to the invention the possibility, the detector in the detection plane, i.e. essentially laterally to the detection direction, to move to images of parts of the object record that composed into an image of the entire object can be.

In a simple, preferred setup no optical elements for guidance the ray paths used. It can but for example mirrors, dichroic mirrors, beam splitters or optical fibers for the leadership of the beam paths used become. Since in the microscope according to the invention the illumination and detection beam paths can be separated the usual use of passive components in other fluorescence microscopes like dichroic mirror or more active, for example acousto-optical Components, for the separation of illuminating and fluorescent light can be dispensed with.

There is a possibility of building for example to be supplemented by a further illuminating light path, the Focused light to a strip or object lighting area which is preferably in the same plane as the object lighting area of the first illuminating light path, so that better illumination the sample is reached. The light for this further illuminating light path can come from the same light source. Advantageously, the Sample from two opposite Directions illuminated. In contrast to 4Pi confocal microscopy [p. Hell and E.H.K. Stelzer, J. Opt. Soc. At the. A 9, 2159 (1992)] the adjustment effort in the microscope according to the invention is low, because there must be two only light strips several micrometers thick are superimposed. In addition, the phase the rays are not taken into account become.

The microscope according to the invention can also operated as a non-confocal 4Pi theta microscope become. The sample is like in a 4Pi (A) confocal microscope illuminated coherently from two opposite directions, so that along this An interference pattern occurs that reflects the intensity in the illumination axis Illumination light plane spatial modulated. This halves the lighting volume, and by shifting the interference pattern (by adjusting the Phase difference between the beams) it is possible to complement each other to illuminate the sample so that an image with increased resolution along the Illumination axis can be reconstructed.

Although it is possible to place the sample on a sample table in the microscope according to the invention or to hold it in air, the sample is preferably held by a holder from above in a water-filled sample chamber and can be rotated about the vertical axis, i.e. in the direction of gravity , This has the advantage that when the sample is rotated to take a picture from a different direction, there is no change in the gravity acting on the sample and it does not deform. With such a rotation of the sample in the sample chamber, the sample chamber is advantageously not moved, so that the optical path lengths (apart from differences due to the refractive index in the sample itself) do not change during the movement process. This leads to better image quality. The sample held in this way can advantageously be aligned in such a way that the influence of strongly scattering or absorbing parts of the sample during image recording is minimized.

It is in another embodiment of the microscope according to the invention also possible, the lighting and detection paths around the fixed object to be examined Object to rotate. Then, however the sample or the object in general to be tracked in further Pictures to be mapped.

The object to be examined is located when shooting in the area-like object lighting area, the object being much larger than is the thickness of this area. A two-dimensional image of the parts of the object located in this area are made by the flat Detector. The object is recorded in three dimensions Rasterization of the object in the direction of detection by the fixed Illumination area (or by scanning the illumination area through the object), with a two-dimensional in each position of the object Picture is taken. The synchronization of movement, lighting and detection is advantageously optimized to the sample load to minimize.

Preferably the rotation of the Object (as well as the linear raster movement) electronically controlled, so that the taking of multiple images from different angles is automated can be and the speed of the sample examination is increased. The number of images and the angle of rotation of the sample taken for a total shot a certain spatial resolution are necessary in favor of a short sample examination time and thus a short one Sample loading can be optimized.

The item to be examined can advantageously be examined Object can also be tilted around the lighting axis so that it still from additional Directions can be observed. In another embodiment of the microscope according to the invention a second detection light path is present which enables the detection of the downward light allowed. Then the object lighting area rotated 90 degrees around the lighting axis (e.g. by the rotation of the cylindrical lens), so the sample can be optically horizontal can be cut (and by a vertical grid movement a three-dimensional image can be created).

Advantageously, in the microscope according to the invention the cylindrical lens is preferably moved at high frequency, for example high-frequency in the illuminating light path along the cylinder axis and / or the lighting axis are moved and / or the cylinder axis is high-frequency be inclined in the direction of the illumination axis, so that the influence of contamination is less strong on the cylindrical lens and the sample is illuminated more evenly becomes.

The bracket can advantageously many biological samples simply by embedding them in a gel (approx. 99% water) can be realized.

By rotating the object to be examined realized recordings from different directions allow one three-dimensional reconstruction of the same through the combination of the individual three-dimensional raw data sets. Because with the preferred embodiment of the microscope according to the invention only a part of the sample can be optimally imaged (in general the two octants that are within the right angle between Illumination and detection axis), are at least four Recordings for a good reconstruction is necessary. These shots can be combine in such a way that the reconstruction offers a higher resolution than the individual ones Recordings. The quality of the reconstructed image by shooting along wider angles so that the blind spots of the common optical transfer function can be filled.

By using lenses with long focal lengths there is a working distance of several millimeters to disposal. The size of the object is primarily due to its translucency limited: provided that the object is completely (and not only the boundary layers) want to examine must be sufficient Light from every part of it in one orientation or another reach the detector.

The invention is illustrated below the attached Drawings closer explained. Show it:

1 the schematic representation of the beam path in an embodiment of the microscope according to the invention, wherein a single illumination beam path and a single detection beam path are present, viewed in the viewing direction I of the 2 ;

2 in the 1 embodiment shown in viewing direction II in 1 ;

3 a schematic diagram of the illumination beam path, which starts from a cylindrical lens and forms an object illumination area in the area of a focus line;

4 a plan view of the beam path of the 3 in viewing direction IV in 3 ;

5 the schematic representation of the Beam path in a further embodiment of the microscope according to the invention, two illumination beam paths being present;

6 another schematic diagram of a microscope according to the invention.

The 1 shows an embodiment of a microscope according to the invention 100 , The structure includes a light source 1 whose collimated light beam 2 through a cylindrical lens 3 in the rehearsal 4 is focused. This creates a thin vertical strip of light 11 , by the in the sample 4 Fluorescence emission can be induced. The light emitted 5 is through detection optics 6 on the flat detector 8th , for example, a CCD camera.

Due to the right-angled arrangement (= 90 degrees) of lighting 9 and detection direction 10 the structure is particularly simple. In particular, dichroic mirrors can be used for the separation of illuminating and fluorescent light in the detection beam path 5 to be dispensed with. The filters 7 in the lighting 2 and in the detection beam path 5 are glass filters or acousto / electro / magneto-optical filters and allow the selective selection of wavelengths for lighting and detection.

The sample 4 is through a bracket 12 in a test chamber 13 held and for image acquisition in the detection direction 10 through the fixed light level 11 emotional. The bracket 12 also allows rotation of the sample 4 about its vertical axis 14 so the sample 4 can be illuminated and observed from several sides.

The 3 and 4 show in principle the previously mentioned and using the cylindrical lens 3 generated illumination beam path 2 , Through the cylindrical lens 3 , whose focal length can preferably be in the range between 10 mm and 100 mm, this is from the light source 1 emitted light focused at a comparatively small angle α. A focus line L thus arises in the 3 Object lighting area shown in dashed lines 20 , which has approximately an area-like or plane-like structure or extension and is formed on both sides of the focus line by cylinder sections. With a dimension a of this object illumination area measured in the direction of the illumination axis or illumination direction 20 of about 5 mm and a thickness dimension of the illumination beam path 2 in the region of the focus line b of approximately 20 μm, the end regions located in the direction of illumination result 22 . 24 of the object lighting area 20 a thickness dimension c of approximately 60 μm, which of course depends on that for the cylindrical lens 3 predefined numerical aperture. Over the entire object lighting area 20 So there is a negligible variation in the thickness of the object lighting area in relation to the dimensions of the objects to be examined 20 in the illumination beam path 2 available, so that, in particular also taking into account the dimensions of the objects to be examined, a thickness of the object illumination area that is constant in a first approximation and thus a flat or planar structure thereof can be assumed.

In the 5 is a modified embodiment of the microscope 100 shown in which two illumination beam paths 2 . 2 ' available. In the case shown, each of these two illumination beam paths 2 . 2 ' , which have mutually opposing directions of illumination, but have mutually corresponding illumination axes, each have a cylindrical lens 3 . 3 ' with associated filter if necessary 7 . 7 ' as well as a light source 1 . 1' on. In a modification of this embodiment, it can further be provided that only a single light source is provided. This results from the superimposition of the two object lighting areas of these lighting beam paths 2 . 2 ' which object lighting areas previously with respect to the 3 and 4 have been described in more detail, a thin vertical light strip, which compared to the light strip in the in 1 illustrated embodiment is more homogeneous. The light emitted 5 is through detection optics 6 on the flat detector 8th displayed. This embodiment of the microscope according to the invention is particularly suitable for absorbent samples in which the entire sample cannot be illuminated with one-sided illumination.

In this embodiment, there is the possibility of the two illumination beam paths 2 . 2 ' or the light rays of the same by defined adjustment of the phase relationship of these light rays to one another where the two object illumination areas of these two illumination beam paths 2 . 2 ' are superimposed on one another to bring about interference. In this way it becomes possible in the area in which the object or sample to be examined 4 To illuminate is to hide certain sections by destructive interference or to highlight certain areas by constructive interference, which can further improve the resolution of the overall system.

In 6 is a further modification of the microscope according to the invention 100 indicated. The arrow P indicates that the cylindrical lens shown there 3 around the illumination axis of the illumination beam path 2 can be rotated, for example by 90 °. This also rotates the object lighting area 20 this illuminating beam path 2 so that starting from the in 2 shown orientation, in which it lies essentially in the plane of the drawing, is rotated by 90 ° and is then perpendicular to the plane of the drawing. In this way it becomes possible to examine the object 4 to be viewed from a different direction, namely that in the representation of the 2 U.N ter this object 4 lying direction. So there can be another detection beam path 5 ' be provided, in which then with respect to the 1 recognizable detection beam path 5 the object to be examined 4 can be viewed at an angle of 90 ° without this object 4 would have been filmed by myself.

With such a system it is possible, for example, using mirrors 60 and a tilting mirror 26 different detection beam paths 5 . 5 ' optionally, depending on the position of the tilting mirror 26 to the same detector 8th or one and the same optical system with lenses 6 to lead. Assigned to the rotational position of the cylindrical lens 3 then becomes the tilting mirror 26 switched accordingly. Of course, it is possible to use two detection beam paths 5 . 5 ' with each assigned lens arrangement and detector independently of one another and to be provided, for example, at an angle of 90 °. Furthermore, it is possible to make at least one of these systems movable, so that it is together with the cylindrical lens 3 around the illumination axis of the illumination beam path 2 in 2 can be rotated so that while rotating the cylindrical lens 3 and this detection beam path then an all-round image of the object to be examined 4 can be created without this object itself being moved.

The invention relates to a microscope, in which a layer of the sample through a thin strip of light 11 is illuminated and the observation takes place perpendicular to the plane of the light strip. The thickness of the strip of light 11 thus essentially determines the depth of field of the system. The object is used for image acquisition 4 due to the fixed light strip with respect to the detector 11 moved, and fluorescence or / and scattered light is recorded with a flat detector. Strongly absorbing or scattering objects 4 are observed from several spatial directions. The three-dimensional images that are taken from any direction can subsequently be combined to form one image in which the data is weighted according to its resolution. The resolution of the combined image is then dominated by the lateral resolution of the individual images.

Claims (15)

Microscope with at least one illumination beam path ( 2 ; 2 . 2 ' ) and at least one detection beam path ( 5 ), characterized in that - with each illumination beam path ( 2 ; 2 . 2 ' ) a focusing arrangement ( 3 ; 3 . 3 ' ) is provided for generating an illumination axis of the illumination beam path ( 2 ; 2 . 2 ' ) and the extensive area-like object lighting area ( 20 ) - that a detection direction ( 10 ) of the at least one detection beam path ( 5 ) approximately orthogonal to the area-like object lighting area ( 20 ), and - that a movement arrangement ( 12 ) is provided to generate a relative movement between the area-like object lighting area ( 20 ) and an object to be examined ( 4 ). Microscope according to claim 1, characterized in that by the movement arrangement ( 12 ) a rotating movement of the object ( 4 ) or / and a moving movement of the object ( 4 ) can be generated. Microscope according to claim 1 or 2, characterized in that the movement arrangement ( 12 ) is designed to provide a substantially fixed area-like object lighting area ( 20 ) the object ( 4 ) to move. Microscope according to claim 1 or 2, characterized in that the movement arrangement is designed for a substantially stationary object ( 4 ) the area-like object lighting area ( 20 ) to move. Microscope according to one of claims 1 to 4, characterized in that the at least one illumination radiation path ( 2 ; 2 . 2 ' ) a cylindrical lens ( 3 ; 3 . 3 ) for focusing the illuminating light. Microscope according to claim 5, characterized in that the cylindrical lens ( 3 ; 3 . 3 ' ) is rotatable about the illumination axis and / or is displaceable in the direction of the illumination or / and the cylinder axis or / and can be tilted with the cylinder axis with respect to the illumination axis. Microscope according to claim 6, characterized in that the movement of the cylindrical lens ( 3 ; 3 . 3 ' ) is a high frequency movement. Microscope according to one of claims 1 to 7, characterized in that that uses scattered light or fluorescent light of one or more wavelengths becomes. Microscope according to one of claims 1 to 8, characterized in that the light source ( 1 ; 1 . 1' ) is a lamp or laser that provides light of one or more wavelengths. Microscope according to one of claims 1 to 9, characterized in that the object ( 4 ) by a bracket ( 12 ) in a sample chamber ( 13 ) is to be held and in it around an axis essentially corresponding to a direction of gravity ( 14 ) is rotatable and movable along at least one direction. Microscope according to one of claims 1 to 10, characterized in that at least two illumination beam paths ( 2 . 2 ' ) with an essentially opposite direction of illumination are provided in order to generate at least regionally overlapping area-like object illumination regions ( 20 ). Microscope according to claim 11, characterized in that the illuminating light of the two illuminating beam paths ( 2 . 2 ' ) in the direction of the lighting axis in the area of the area-like object lighting area ( 20 ) interferes at least in certain areas. Microscope according to claim 12, characterized in that the illuminating light of the two illuminating beam paths ( 2 . 2 ' ) has a constant, adjustable phase. Microscope according to one of claims 1 to 13, characterized in that the at least one detection beam path ( 5 ) has a detector and that the detector laterally with respect to the detection direction of the at least one detection beam path ( 5 ) is movable. Microscope according to one of claims 1 to 14, characterized in that the at least one detection beam path ( 5 ; 5 ' ) is adaptable in such a way that the detection direction when the object lighting area is shifted ( 20 ) approximately orthogonal to the area-like object lighting area ( 20 ) lies.